Gut Microbiota Depletion Using Antibiotics to Investigate Diet-Derived Microbial Metabolites: An Efficient Strategy.

SCOPE: Gut microbiota depletion using antibiotics in drinking water is a valuable tool to investigate the role of gut microbes and microbial metabolites in health and disease. However, there are challenges associated with this model. Animals avoid drinking water because of the antibiotic bitterness, which affects their metabolic health. The present study develops an efficient strategy to deplete gut microbes without affecting metabolic parameters. METHODS AND RESULTS: Male C57BL/6J mice (7 weeks old) are fed a control (C) or high-fat (HF) diet. Subgroups of C and HF mice receive an antibiotic cocktail in drinking water (CA and HA). The antibiotic dosage is gradually increased so that the animals adapt to the taste of antibiotics. Metabolic parameters, gut microbiome, and microbial metabolites are assessed after 12 weeks treatment. Culture methods and 16s rRNA amplification confirm the depletion of gut microbes in antibiotic groups (CA and HA). Further, antibiotic treatment does not alter metabolic parameters (body weight, body fat, lean body mass, blood glucose, and glucose/insulin tolerance), whereas it suppresses the production of diet-derived microbial metabolites (trimethylamine and trimethylamine-N-oxide). CONCLUSION: This strategy effectively depletes gut microbes and suppresses the production of microbial metabolites in mice without affecting their metabolic health.

Blueberry intervention mitigates detrimental microbial metabolite trimethylamine N-oxide by modulating gut microbes.

Gut microbes play a pivotal role in host physiology by producing beneficial or detrimental metabolites. Gut bacteria metabolize dietary choline and L-carnitine to trimethylamine (TMA) which is then converted to trimethylamine-N-oxide (TMAO). An elevated circulating TMAO is associated with diabetes, obesity, cardiovascular disease, and cancer in humans. In the present study, we investigated the effect of dietary blueberries and strawberries at a nutritional dosage on TMA/TMAO production and the possible role of gut microbes. Blueberry cohort mice received a control (C) or freeze-dried blueberry supplemented (CB) diet for 12 weeks and subgroups received an antibiotics cocktail (CA and CBA). Strawberry cohort mice received a control (N) or strawberry-supplemented (NS) diet and subgroups received antibiotics (NA and NSA). Metabolic parameters, choline, TMA, and TMAO were assessed in addition to microbial profiling and characterization of berry powders. Blueberry supplementation (equivalent to 1.5 human servings) reduced circulating TMAO in CB versus C mice (~48%) without changing choline or TMA. This effect was not mediated through alterations in metabolic parameters. Dietary strawberries did not reduce choline, TMA, or TMAO. Depleting gut microbes with antibiotics in these cohorts drastically reduced TMA and TMAO to not-quantified levels. Further, dietary blueberries increased the abundance of bacterial taxa that are negatively associated with circulating TMA/TMAO suggesting the role of gut microbes. Our phenolic profiling indicates that this effect could be due to chlorogenic acid and increased phenolic contents in blueberries. Our study provides evidence for considering dietary blueberries to reduce TMAO and prevent TMAO-induced complications.

Dose- and Time-Dependent Effect of Dietary Blueberries on Diabetic Vasculature Is Correlated with Gut Microbial Signature.

Evidence from our lab and others indicates the vascular effects of dietary blueberries. In the present study, we determined dietary blueberries' dose- and time-dependent effects on diabetic vasculature and their association with gut microbes. Seven-week-old db/db diabetic male mice were fed a diet supplemented with ± freeze-dried wild blueberry powder (FD-BB) for 4, 8, or 12 weeks (three cohorts). Diets contained 0%, 1.23%, 2.46%, and 3.7% of FD-BB, equivalent to 0, ½, 1, and 1.5 human servings of wild blueberries, respectively. The non-diabetic db/+ mice fed a standard diet served as controls. Metabolic parameters, vascular inflammation, and gut microbiome were assessed. Dietary supplementation of 3.7% FD-BB improved vascular inflammation in diabetic mice without improving systemic milieu in all three cohorts. Blueberries improved diabetes-induced gut dysbiosis depending on blueberry dosage and treatment duration. Spearman's correlation indicated that the opportunistic microbes and commensal microbes were positively and negatively associated with indices of vascular inflammation, respectively. Dietary blueberries reduced the opportunistic microbe that was positively associated with vascular inflammation (Desulfovibrio), and increased the commensal microbe that was negatively associated with vascular inflammation (Akkermansia). Dietary blueberries could be a potential adjunct strategy to beneficially modulate gut microbes and improve vascular complications in diabetes.

Gut Microbes Are Associated with the Vascular Beneficial Effects of Dietary Strawberry on Metabolic Syndrome-Induced Vascular Inflammation.

SCOPE: Metabolic syndrome (MetS) alters the gut microbial ecology and increases the risk of cardiovascular disease. This study investigates whether strawberry consumption reduces vascular complications in an animal model of MetS and identifies whether this effect is associated with changes in the composition of gut microbes. METHODS AND RESULTS: Seven-week-old male mice consume diets with 10% (C) or 60% kcal from fat (high-fat diet fed mice; HF) for 12 weeks and subgroups are fed a 2.35% freeze-dried strawberry supplemented diet (C+SB or HF+SB). This nutritional dose is equivalent to ≈160 g of strawberry. After 12 weeks treatment, vascular inflammation is enhanced in HF versus C mice as shown by an increased monocyte binding to vasculature, elevated serum chemokines, and increased mRNA expression of inflammatory molecules. However, strawberry supplementation suppresses vascular inflammation in HF+SB versus HF mice. Metabolic variables, blood pressure, and indices of vascular function were similar among the groups. Further, the abundance of opportunistic microbe is decreased in HF+SB. Importantly, circulating chemokines are positively associated with opportunistic microbes and negatively associated with the commensal microbes (Bifidobacterium and Facalibaculum). CONCLUSION: Dietary strawberry decreases the abundance of opportunistic microbe and this is associated with a decrease in vascular inflammation resulting from MetS.

Dietary Blueberry Ameliorates Vascular Complications in Diabetic Mice Possibly through NOX4 and Modulates Composition and Functional Diversity of Gut Microbes.

SCOPE: In diabetes, endothelial inflammation and dysfunction play a pivotal role in the development of vascular disease. This study investigates the effect of dietary blueberries on vascular complications and gut microbiome in diabetic mice. METHODS AND RESULTS: Seven-week-old diabetic db/db mice consume a standard diet (db/db) or a diet supplemented with 3.8% freeze-dried blueberry (db/db+BB) for 10 weeks. Control db/+ mice are fed a standard diet (db/+). Vascular inflammation is assessed by measuring monocyte binding to vasculature and inflammatory markers. Isometric tension procedures are used to assess mesenteric artery function. db/db mice exhibit enhanced vascular inflammation and reduced endothelial-dependent vasorelaxation as compared to db/+ mice, but these are improved in db/db+BB mice. Blueberry supplementation reduces the expression of NOX4 and IκKß in the aortic vessel and vascular endothelial cells (ECs) isolated from db/db+BB compared to db/db mice. The blueberry metabolites serum reduces glucose and palmitate induced endothelial inflammation in mouse aortic ECs. Further, blueberry supplementation increases commensal microbes and modulates the functional potential of gut microbes in diabetic mice. CONCLUSION: Dietary blueberry suppresses vascular inflammation, attenuates arterial endothelial dysfunction, and supports the growth of commensal microbes in diabetic mice. The endothelial-specific vascular benefits of blueberries are mediated through NOX4 signaling.

Dietary supplementation with strawberry induces marked changes in the composition and functional potential of the gut microbiome in diabetic mice.

Gut microbiota contributes to the biological activities of berry anthocyanins by transforming them into bioactive metabolites, and anthocyanins support the growth of specific bacteria, indicating a two-way relationship between anthocyanins and microbiota. In the present study, we tested the hypothesis that strawberry supplementation alters gut microbial ecology in diabetic db/db mice. Control (db/+) and diabetic (db/db) mice (7 weeks old) consumed standard diet or diet supplemented with 2.35% freeze-dried strawberry (db/db + SB) for 10 weeks. Colon contents were used to isolate bacterial DNA. V4 variable region of 16S rRNA gene was amplified. Data analyses were performed using standardized pipelines (QIIME 1.9 and R packages). Differences in predictive metagenomics function were identified by PICRUSt. Principal coordinate analyses confirmed that the microbial composition was significantly influenced by both host genotype and strawberry consumption. Further, α-diversity indices and ß-diversity were different at the phylum and genus levels, and genus and operational taxonomical units levels, respectively (P<.05). At the phylum level, strawberry supplementation decreased the abundance of Verrucomicrobia in db/db + SB vs. db/db mice (P<.05). At the genus level, db/db mice exhibited a decrease in the abundance of Bifidobacterium, and strawberry supplementation increased Bifidobacterium in db/db + SB vs. db/db mice (P<.05). PICRUSt revealed significant differences in 45 predicted metabolic functions among the 3 groups. Our study provides evidence for marked changes in the composition and functional potential of the gut microbiome with strawberry supplementation in diabetic mice. Importantly, strawberry supplementation increased the abundance of beneficial bacteria Bifidobacterium which play a pivotal role in the metabolism of anthocyanins.

Circulating metabolites of strawberry mediate reductions in vascular inflammation and endothelial dysfunction in db/db mice.

BACKGROUND: Cardiovascular disease is 2-4-fold more prevalent in patients with diabetes. Human studies support the cardiovascular benefits of strawberry consumption but the effects of strawberry on diabetic vasculature are unknown. We tested the hypothesis that dietary strawberry supplementation attenuates vascular inflammation and dysfunction in diabetic mice. METHODS: Seven-week-old diabetic db/db mice that consumed standard diet (db/db) or diet supplemented with 2.35% freeze-dried strawberry (db/db + SB) for ten weeks were compared to non-diabetic control mice (db/+). Indices of vascular inflammation and dysfunction were measured. Endothelial cells (ECs) were isolated from the vasculature to determine the influence of strawberry on them. The effect of metabolites of strawberry on endothelial inflammation was determined by incubating mouse aortic ECs (MAECs) with ±5% serum, obtained from strawberry fed mice (metabolites serum) or standard diet fed mice (control serum) ±â€¯25 mM glucose and 100 µM palmitate. RESULTS: db/db mice exhibited an increased monocyte binding to vessel, elevated blood pressure, and reduced endothelial-dependent vasorelaxation compared with db/+ mice but each defect was attenuated in db/db + SB mice. The elevation of inflammatory molecules, NOX2 and inhibitor-κB kinase observed in ECs from db/db vs. db/+ mice was suppressed in db/db + SB mice. Glucose and palmitate increased endothelial inflammation in MAECs but were normalized by co-incubation with metabolites serum. CONCLUSIONS: Dietary supplementation of strawberry attenuates indices of vascular inflammation and dysfunction in diabetic db/db mice. The effect of strawberry on vasculature is endothelial-dependent and possibly mediated through their circulating metabolites. Strawberry might complement conventional therapies to improve vascular complications in diabetics.

Blueberry Metabolites Attenuate Lipotoxicity-Induced Endothelial Dysfunction.

SCOPE: Lipotoxicity-induced endothelial dysfunction is an important vascular complication associated with diabetes. Clinical studies support the vascular benefits of blueberry anthocyanins, but the underlying mechanism is unclear. The hypothesis that metabolites of blueberry anthocyanins attenuate lipotoxicity-induced endothelial dysfunction was tested. METHODS AND RESULTS: Human aortic endothelial cells (HAECs) were treated for 6 h with either: (i) the parent anthocyanins (malvidin-3-glucoside and cyanidin-3-glucoside); or (ii) the blueberry metabolites (hydroxyhippuric acid, hippuric acid, benzoic acid-4-sulfate, isovanillic acid-3-sulfate, and vanillic acid-4-sulfate), at concentrations known to circulate in humans following blueberry consumption. For the last 5 h HAECs were treated with palmitate or vehicle. HAECs treated with palmitate displayed elevated reactive oxygen species generation, increased mRNA expression of NOX4, chemokines, adhesion molecules, and IκBα, exaggerated monocyte binding, and suppressed nitric oxide production. Of note, the damaging effects of palmitate were ameliorated in HAECs treated with blueberry metabolites but not parent anthocyanins. Further, important translational relevance of these results was provided by our observation that palmitate-induced endothelial dysfunction was lessened in arterial segments that incubated concurrently with blueberry metabolites. CONCLUSION: The presented findings indicate that the vascular benefits of blueberry anthocyanins are mediated by their metabolites. Blueberries might complement existing therapies to lessen vascular complications.

Mechanistic insights into the vascular effects of blueberries: Evidence from recent studies.

Cardiovascular disease is the leading cause of death in the United States. Dietary habits influence a variety of cardiovascular complications such as peripheral artery disease, heart failure, and kidney disease. We along with others have previously reported the cardiovascular beneficial effects of dietary flavonoids. Anthocyanins, one class of flavonoids widely available in berries, have recently drawn wide scientific attention because of their diverse health benefits. Epidemiological, clinical, and animal studies indicate that blueberry anthocyanins exert protection against cardiovascular complications by acting on multiple targets in the vascular system. These include activating endothelial nitric oxide synthase signaling, reducing oxidative stress, improving inflammatory pathways, and ameliorating dyslipidemia. Anthocyanins are extensively metabolized in humans suggesting that their vascular benefits are likely mediated by their circulating metabolites. However, the bioactivities of blueberry metabolites are unknown. Evaluating the bioactivities of metabolites, analyzing their structure-activity relationship, and well-designed human trials are needed to understand the potential vascular effects of blueberries and their metabolites. Understanding the vascular effects will provide a solid scientific foundation to recommend blueberries to improve vascular health. This review highlights the recent developments in the understanding of the vascular effects of blueberries with special emphasis on the molecular mechanisms involved.